JP6472488B2 - Bioassay and measurement kit for antibodies to thyroid stimulating hormone receptor and novel genetically modified cells used in these - Google Patents

Description

  The present invention relates to a thyroid stimulating hormone receptor (TSHR), a cell expressing a cAMP-dependent calcium channel and a calcium-sensitive protein, a composition containing the cell, use of the composition for thyroid disease diagnosis, and use of the composition The present invention relates to a method for diagnosing thyroid diseases.

Thyroid-stimulating hormone (TSH) produced by the pituitary gland binds to the thyroid-stimulating hormone receptor (TSHR) present in the thyroid gland and promotes the secretion of thyroid hormone. Thyroid hormone is a hormone that enhances the metabolism of the whole body. Therefore, abnormally increased or decreased action of this hormone has various effects on the mind and body, causing thyroid disease. For example, in Graves' disease, a TSHR-stimulating antibody (TSAb) is produced in the body, and it stimulates TSHR excessively instead of TSH, so that thyroid function is enhanced, thyroid enlargement, eyeball protrusion Symptoms such as tachycardia appear. On the other hand, in hypothyroidism, TSHR binding inhibitory antibody (TSBAb: Thyroid stimulation blocking antibody) is produced, thyroid function is lowered, and symptoms such as weight gain, depression, and general fatigue appear. Is also present.
Until now, measurement of these autoantibodies (TSAb and TSBAb) in blood has been used for diagnosis of Graves' disease and hypothyroidism.
As a typical measurement method, a radioreceptor assay method is used in which a radioisotope-labeled monoclonal antibody against TSH or TSHR is used, and the amount of bound antibody is measured by competitively inhibiting the binding between autoantibodies and TSHR in patient serum ( TBII method) or a bioassay method for measuring the amount of TSAb by allowing an antibody that binds to TSHR to act on porcine thyroid cells and the like, and measuring the increase in the concentration of cAMP in thyroid cells using radioisotope-labeled cAMP (TSAb method) has been reported (Non-patent document 1 and Non-patent document 2).
Methods in Enzymology, 74, 405-420 (1981) J Clin Endocrinol Metab. 1986 May; 62 (5): 855-62

  The object of the present invention is to provide a composition for measuring TSAb and / or TSBAb, a composition for diagnosing thyroid disease, etc., which can be operated more easily than conventional methods without using radioisotopes requiring special techniques and equipment. Is to provide. A further object of the present invention is to provide a method for diagnosing thyroid disease using the composition.

  The present inventor used the calcium-sensitive protein to determine the amount of calcium ion influx into the cell caused by cAMP produced by binding of a TSHR stimulating antibody (TSAb) to a thyroid stimulating hormone receptor (TSHR). It was measured. Thereby, the present inventor succeeded in measuring the amount of TSAb without using a radioisotope. Furthermore, using the same principle, the present inventors have succeeded in measuring the amount of TSHR binding inhibitory antibody (TSBAb), thereby completing the present invention.

That is, the present invention relates to cells expressing thyroid stimulating hormone receptor (TSHR), cAMP-dependent calcium channel, and calcium-sensitive protein, and compositions and kits containing the cells.
The present invention also provides a thyroid gland for measuring the amount of TSHR-stimulating antibody and / or TSHR-binding-inhibiting antibody in a biological sample, including cells expressing TSHR, cAMP-dependent calcium channel, and calcium-sensitive protein. The present invention relates to a composition and kit for diagnosing a disease, for determining a human at high risk of developing a thyroid disease, or for determining a therapeutic effect of a human undergoing treatment for a thyroid disease.
Furthermore, the present invention provides the following steps (1), (2) and (3) or (1 ′), (2) and (3):
(1) Thyroid-stimulating hormone receptor (TSHR), cAMP-dependent calcium channel, and cells expressing calcium-sensitive protein, luminescent substrate of calcium-sensitive protein, Ca ²⁺ -free medium or Ca ²⁺ and Mg ²⁺ free A mixture comprising a sample of the medium, TSH and a blood sample of the subject; or (1 ′) a thyroid stimulating hormone receptor (TSHR), a cAMP-dependent calcium channel, and a cell expressing a calcium sensitive protein, a calcium sensitive protein Preparing a mixture comprising a luminescent substrate, Ca ²⁺ free medium or Ca ²⁺ and Mg ²⁺ free medium and a sample from the blood of the subject;
(2) adding a Ca ²⁺ -containing solution to the mixture prepared in (1) or (1 ′); and (3) measuring luminescence of calcium-sensitive protein generated from the cells.
A method for determining a thyroid disease, a method for determining a human at high risk of developing a thyroid disease, or a method for determining the effectiveness of a treatment for a thyroid disease.
Furthermore, the present invention provides the following steps (1) to (3):
(1) Thyroid-stimulating hormone receptor (TSHR), cAMP-dependent calcium channel, and a cell containing a calcium-sensitive protein, a luminescent substrate for calcium-sensitive protein, a medium containing Ca ²⁺ , TSH and a sample derived from the blood of a subject To prepare;
(2) adding a forskolin-containing solution to the mixture prepared in (1); and
(3) Measure the luminescence of calcium sensitive protein generated from the cells,
A method for determining hypothyroidism, a method for determining a human at high risk of developing hypothyroidism, or a method for determining the effectiveness of a treatment for hypothyroidism.

  According to the present invention, the amount of TSHR-stimulating antibody (TSAb) in a biological sample and the TSHR binding-inhibiting antibody (TSBAb) can be easily and safely operated without requiring complicated operations associated with the use of radioisotopes. ) Can be measured and thyroid disease can be diagnosed.

It is a figure which shows that the light emission emitted from the CHO cell line which expresses human TSHR, a modified CNG channel, and a modified aequorin is dependent on the density | concentration of bovine origin TSH (bTSH). It is a figure which shows the light-emission quantity emitted from the CHO cell line which expresses human TSHR, a modified CNG channel, and a modified aequorin in low dose bTSH. It is a figure which shows the influence of the density | concentration of an aequorin luminescent substrate, and culture | cultivation time with respect to the light-emission amount emitted from the CHO cell strain which expresses human TSHR, a modified CNG channel, and a modified aequorin. It is a figure which shows the influence of the TSHR expression plasmid amount to introduce | transduce with respect to the light-emission amount emitted from the CHO cell line which expresses human TSHR, a modified CNG channel, and a modified aequorin. It is a figure which shows the relationship between the cell density | concentration of the CHO cell line which expresses human TSHR, a modified CNG channel, and a modified aequorin, and luminescence amount. It is the figure which represented the relationship between the cell density | concentration and light-emission quantity of the CHO cell line which expresses human TSHR, a modified CNG channel, and a modified aequorin by the relative value which set each blank value to 1. Human TSHR, for the light emission amount emitted from the CHO cell line expressing a modified CNG channels and modified aequorin shows the effect of the concentration of CaCl ₂ to be added. It is a figure which shows that the kit which concerns on this invention can quantify TSHR stimulation type | mold antibody (TSAb). It is a figure which shows that the kit which concerns on this invention can detect a TSHR binding inhibition antibody (TSBAb). It is a figure which shows that the kit which concerns on this invention can detect a TSHR binding inhibition type antibody (TSBAb) using the desensitization of a cell. It is a figure which shows that the kit which concerns on this invention can detect a TSHR stimulation type | mold antibody (TSAb) with a higher sensitivity than the conventional product (Thyroid stimulation autoantibody kit TSAb kit "Yamasa" (trademark)). It is a figure which shows the time-dependent change of the light-emission quantity emitted from the CHO cell line which expresses human TSHR, a modified CNG channel, and a modified aequorin. It is a figure which shows the density | concentration dependence of an inhibitory antibody. It is a figure which shows the density | concentration dependence of the detection of the inhibitory antibody by forskolin solution addition. FIG. 3 is a graph showing changes in the amount of luminescence with the incubation time of a stimulating antibody (TSAb). FIG. 6 shows changes in the amount of luminescence with the incubation time of an inhibitory antibody (TSBAb). It is the figure which showed the change of the light-emission quantity by the culture | cultivation time of the inhibitory antibody (TSBAb) by forskolin addition. FIG. 6 shows plasmid pmCNGα2. FIG. 3 shows plasmid pcDNA mt s AEQ. It is a figure which shows the histogram of the TSAb value of 48 healthy persons measured using the kit based on this invention. It is a figure which shows distribution of the TSAb value in the serum sample derived from various thyroid diseases measured using the kit which concerns on this invention.

  The present invention provides cells that express thyroid stimulating hormone receptor (TSHR), cAMP-dependent calcium channels, and calcium-sensitive proteins, and compositions comprising the cells.

The thyroid stimulating hormone receptor (TSHR) may be any receptor to which thyroid stimulating hormone (TSH) binds, and includes a receptor that activates adenylate cyclase to increase cAMP. The origin of TSHR is not particularly limited as long as it is a mammal, and can be, for example, human, mouse, cow, rat, or pig. TSHR may have one or several amino acids modified (addition, substitution, deletion, etc.) in its amino acid sequence as appropriate, or TSHR has an amino acid sequence that is different from that of natural TSHR. A protein comprising an amino acid sequence having a homology of 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more, wherein TSH binds to adenylate cyclase It may be a protein having a function of activating the cAMP to increase cAMP.
TSHR can be a protein having the amino acid sequence set forth in SEQ ID NO: 1. Further, TSHR is an amino acid sequence having a homology with the amino acid sequence shown in SEQ ID NO: 1 of 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more A protein having a function of binding TSH and activating adenylate cyclase to increase cAMP.
Alternatively, TSHR can be a chimeric protein of TSHR with receptors related to TSHR, such as lutein hormone receptor, follicle stimulating hormone receptor, human chorionic gonadotropin receptor, and the like. These chimeric proteins are made by replacing portions other than amino acid residues 8-89 or 8-165 of TSHR with appropriate portions of the lutein hormone receptor, follicle stimulating hormone receptor, human chorionic gonadotropin receptor. May be. For example, to create a TSHR-lutein hormone receptor chimeric protein, amino acid residues 90-165 of TSHR are replaced with segment Mc2 of the LH-CG receptor, and amino acid residues 261-370 of TSHR are further replaced with LH- It may be replaced with segment Mc4 of CG receptor.
In the present invention, TSH is not particularly limited as long as it is derived from a mammal. For example, TSH may be derived from human, mouse, cow, rat, or pig. As for TSH, one or several amino acids may be modified (addition, substitution, deletion, etc.) in its amino acid sequence, and the homology of amino acid sequence with respect to natural TSH is 70% or more. 80% or higher, 85% or higher, 90% or higher, 95% or higher, 98% or higher, or 99% or higher amino acid sequence that binds to TSHR and activates adenylate cyclase to increase cAMP It may be a protein having the function of

The cAMP-dependent calcium channel is a channel that changes the inflow amount of calcium ions into cells in response to a change in cAMP concentration, and a channel that increases the inflow amount of calcium ions into cells in response to an increase in cAMP concentration. including. An example of a cAMP-dependent calcium channel is a CNG (cyclic nucleotide gated ion channel) calcium channel. In some cases, one or several amino acids may be modified (addition, substitution, deletion, etc.) in the amino acid sequence. For example, modification (substitution, addition, Deletion). A CNG calcium channel has a homology of 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more with respect to a natural CNG calcium channel. There may be a protein consisting of an amino acid sequence, which increases the inflow of calcium ions into cells in response to an increase in cAMP concentration. Examples of modifications include substitution of 460th cysteine with tryptophan in murine CNG calcium channel, substitution of 583th glutamic acid with methionine in murine CNG calcium channel, serine, methionine of 537th threonine in bovine CNG calcium channel, Examples include substitution to valine or alanine, and combinations thereof. The substitutions listed above are not limited to the animal species from which they are derived, but also apply to amino acid substitutions at corresponding sites in other animal species. For example, the threonine of the mouse CNG calcium channel corresponding to the 537th threonine in the bovine CNG calcium channel can be substituted with serine, methionine, valine or alanine. The substitution can be performed at one or more positions. For example, substitution of 460th cysteine with tryptophan in the mouse CNG calcium channel and substitution of 583th glutamic acid with methionine can also be performed.
CNG calcium channels can consist of α-subunits and / or β-subunits. The configuration thereof is arbitrary, and for example, it can be configured of at least one subunit selected from the group consisting of an α2 subunit, an α3 subunit, an α4 subunit, and a β1b subunit. Each subunit can then be modified as described above.
The origin of the CNG calcium channel is not particularly limited as long as it is a mammal, and may be, for example, human, mouse, cow, rat, or pig. The CNG calcium channel can be a protein having the amino acid sequence shown in SEQ ID NO: 2. Furthermore, the CNG calcium channel has a homology with the amino acid sequence shown in SEQ ID NO: 2 of 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more. It is a protein consisting of an amino acid sequence, and may be a protein that increases the amount of calcium ion inflow into cells in response to an increase in cAMP concentration.

Calcium-sensitive proteins include proteins whose structure changes in response to calcium, and include proteins that sense calcium and emit light, that is, proteins that function as so-called calcium sensors.
Examples of calcium-sensitive proteins include aequorin, cameleon (Invitrogen), Case12 (Evrogne), Krytin, Oberin, Mytrocomin, Minopsin, Belvoine, calcium-sensitive calmodulin and one of the myosin light chain kinases that bind to it. A protein that binds GFP of two colors different from the sequence of the region, a calcium-sensitive protein that binds calmodulin between amino acids 144 and 146 of the amino acid sequence of GFP, and probe number G3 described in JP-A-2002-153279 -85, A1-2 proteins or, if present, these apoproteins (eg, apoaequorin).
The calcium-sensitive protein may have its amino acid sequence modified (addition, substitution, deletion, etc.) as appropriate according to the purpose, and may be modified to increase the amount of luminescence and / or improve the SN ratio. Modifications include the addition, substitution or deletion of one or several amino acids in the amino acid sequence. Calcium sensitive proteins have amino acids that are 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more or 99% or more homologous to the amino acid sequence of a natural calcium sensitive protein A protein comprising a sequence that emits light upon sensing calcium is included. For example, a calcium sensitive protein can be modified to optimize its gene for human codon frequency and have a mitochondrial translocation signal.
The calcium sensitive protein can be a protein having the amino acid sequence shown in SEQ ID NO: 3. Furthermore, the calcium sensitive protein has a homology with the amino acid sequence shown in SEQ ID NO: 3 of 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more. It is a protein consisting of an amino acid sequence and can be a protein that senses calcium and emits light.

In the cells expressing thyroid stimulating hormone receptor (TSHR), cAMP-dependent calcium channel, and calcium-sensitive protein according to the present invention, thyroid-stimulating hormone receptor (TSHR), cAMP-dependent calcium channel, and calcium-sensitive protein are respectively Expressed transiently or stably in cells. The cells are not particularly limited, and may be cell lines such as CHO cells, HEK293 cells, and 3T3 cells. For example, a cell expressing a thyroid stimulating hormone receptor (TSHR), a cAMP-dependent calcium channel, and a calcium-sensitive protein according to the present invention has TSHR having the amino acid sequence represented by SEQ ID NO: 1, and the cAMP-dependent calcium channel is A CHO cell having a modified CNG calcium channel having the amino acid sequence represented by SEQ ID NO: 2, wherein the calcium sensitive protein is a modified apoaequorin having the amino acid sequence represented by SEQ ID NO: 3, and each protein is stably expressed It can be. Furthermore, for example, cells expressing the thyroid stimulating hormone receptor (TSHR), cAMP-dependent calcium channel, and calcium-sensitive protein according to the present invention have a TSHR, a cAMP-dependent calcium channel, and a calcium-sensitive protein, respectively. A protein comprising an amino acid sequence having homology of 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more with respect to any one amino acid sequence of -3 Each can be a CHO cell stably expressing a protein that retains the function of a thyroid stimulating hormone receptor (TSHR), a cAMP-dependent calcium channel, or a calcium sensitive protein.
A cell that naturally expresses one or more proteins selected from the group consisting of thyroid stimulating hormone receptor (TSHR), cAMP-dependent calcium channel, and calcium-sensitive protein may be used, but not expressed in the cell. Cells expressing the thyroid stimulating hormone receptor (TSHR), cAMP-dependent calcium channel, and calcium-sensitive protein according to the present invention can be prepared by expressing the protein transiently or stably. As an example of this, cAMP-dependent calcium channels and calcium-sensitive proteins can be transiently or stably added to thyroid-derived cells that endogenously express TSHR, such as FRTL-5 or Nthy-ori 3-1, respectively. And cells in which TSHR and calcium-sensitive protein are transiently or stably forcibly expressed in cells derived from olfactory tissue that endogenously express CNG calcium channels.

  Cells expressing the thyroid stimulating hormone receptor (TSHR), cAMP-dependent calcium channel, and calcium-sensitive protein according to the present invention can be cryopreserved. Cryopreservation can be stored in a cell cryopreservation solution at an appropriate temperature, such as -20 ° C or -80 ° C. The cell cryopreservation solution is not limited, but Cell Bunker (registered trademark) (Nippon Zenyaku Kogyo Co., Ltd.), Bunker (registered trademark) (Lymphtec Corporation), Cellvation (registered trademark) (CELOX LABORATORIES, Inc.) , CryoStor (registered trademark) (BIOLIFE SOLUTIONS) and the like. By cryopreserving a large number of cells of the same lot, measurement errors derived from cells between compositions or kits can be greatly suppressed, and the reproducibility of measurement results can be improved. . In addition, cells expressing the thyroid stimulating hormone receptor (TSHR), cAMP-dependent calcium channel, and calcium-sensitive protein according to the present invention are present in human blood even after being cryopreserved and thawed in a warm bath or the like. Retains sufficient sensitivity to detect TSHR-stimulated antibodies (TSAb) and TSHR binding inhibitory antibodies (TSBAb). In addition, after seeding in a suitable container after thawing and culturing for about 2 hours, the state of the cells is deteriorated by the reagents added for detection of TSAb and TSBAb and components derived from human blood. Absent.

Thyroid-stimulating hormone receptor (TSHR) according to the present invention, cAMP-dependent calcium channel, and a composition comprising cells expressing a calcium-sensitive protein (for example, thyroid-stimulating hormone receptor (TSHR) according to the present invention, cAMP-dependent calcium channel) And an aqueous solution containing cells expressing calcium-sensitive protein) for measuring the amount of TSHR-stimulating antibody and / or the amount of TSHR binding-inhibiting antibody, for diagnosing thyroid disease, and risk of developing thyroid disease It can be used for the determination of high humans and / or for the determination of the therapeutic effect of humans undergoing treatment for thyroid diseases.
In the present specification, thyroid diseases include hyperthyroidism and hypothyroidism, hyperthyroidism includes Graves' disease, and hypothyroidism includes Hashimoto's disease. In this specification, Hashimoto's disease includes hypothyroidism in which TSBAb is positive in the blood, atrophic thyroiditis without goiter, atrophic thyroiditis caused by TSBAb, and myxedema.
The cells expressing thyroid stimulating hormone receptor (TSHR), cAMP-dependent calcium channel, and calcium-sensitive protein are as described above.
TSHR stimulating antibodies (TSAbs) are antibodies that can act as agonists of TSHR and can be found in the blood of Graves' disease patients.
A TSHR binding inhibitory antibody (TSBAb) is an antibody that binds to TSHR and can act as an antagonist of TSHR, and includes, for example, an antibody that competitively inhibits binding of TSH to TSHR. TSHR binding inhibitory antibodies (TSBAb) can be found in the blood of patients with hypothyroidism, for example, in the blood of patients with Hashimoto's disease.

A TSHR stimulating antibody (TSAb) or a TSHR binding inhibitory antibody (TSBAb) present in a biological sample can be measured using the following mechanism of action.
(1) When a TSHR-stimulating antibody (TSAb) is present in a biological sample, TSAb increases cAMP by acting on TSHR on cells according to the present invention. As a result, the CNG calcium channel is activated and the intracellular influx of calcium increases, so that the calcium sensitive protein emits light. That is, the presence of TSAb in the sample appears as an output of luminescence of calcium sensitive protein.
(2) When a TSHR binding inhibitory antibody (TSBAb) is present in a biological sample (a) A method using competitive inhibition with TSH TSBAb is added together with TSH to bind TSH to TSHR. Competitive inhibition. Thereby, by inhibiting the action of TSH and suppressing an increase in the concentration of cAMP, an increase in the influx of calcium via the CNG calcium channel is also suppressed, and the luminescence of the calcium sensitive protein is suppressed. That is, the presence of TSBAb in the sample appears as an output of suppression of luminescence of the calcium sensitive protein.
(B) Method utilizing desensitization of CNG calcium channel TSBAb is added together with TSH to competitively inhibit the binding of TSH to TSHR. Thereby, the effect | action of TSH is inhibited and the increase in the density | concentration of cAMP is suppressed. Here, if TSBAb is not present, the concentration of cAMP is increased by the action of TSH and the CNG calcium channel is activated, but after a certain time, the CNG calcium channel is desensitized and newly added. Does not respond to forskolin (or agents that increase the concentration of cAMP). Therefore, if TSBAb is not present in the sample, calcium does not enter the cell, and appears as an output of suppression of luminescence of the calcium sensitive protein. On the other hand, if TSBAb is present in the sample, the CNG calcium channel is not desensitized and the luminescence of the calcium sensitive protein is not suppressed.

In one embodiment of the present invention, a TSHR-stimulating antibody and / or a TSHR binding-inhibiting antibody is detected in a biological sample by using the above-described mechanism of action and using the composition according to the present invention. The concentration of TSHR stimulating antibodies and / or TSHR binding inhibiting antibodies in the two biological samples can be compared, or TSHR stimulating antibodies and / or TSHR binding inhibition in the two biological samples can be compared The relative amount of antibody of the type can be measured. Furthermore, by using the composition according to the present invention, the concentration of a TSHR stimulating antibody and / or a TSHR binding inhibitory antibody in a biological sample can be measured.
In this specification, biological samples include samples derived from living organisms such as blood and samples prepared from blood, for example, human blood, samples prepared from human blood, dog blood, and dog blood. Samples prepared, cat blood, samples prepared from cat blood are included.

Since the composition according to the present invention can be used to measure TSHR-stimulating antibodies and / or TSHR binding-inhibiting antibodies in human blood, it is possible to diagnose whether or not a subject suffers from thyroid disease. it can.
When the action mechanism described in (1) above is used, Graves' disease can be diagnosed using the composition according to the present invention. For example, the amount of luminescence of calcium sensitive protein emitted from cells added with a blood sample of a subject and the amount of luminescence of calcium sensitive protein emitted from cells added with a normal blood sample (standard) of the same amount as the blood sample of the subject By measuring the amount, Graves' disease can be diagnosed. In this case, if the amount of luminescence emitted from the cells to which the blood sample of the subject is added is higher than the amount of luminescence emitted from the cells to which the blood sample (standard product) of a normal person is added, a TSHR stimulating antibody in the blood of the subject It can be judged that the concentration of (TSAb) is higher than that of a normal person, and the subject can be diagnosed as having Graves' disease.
In addition, the amount of luminescence (integrated value) emitted from a large number of normal human populations, for example, cells to which 50 to 100 normal human blood samples have been added is measured in advance, and the average value and standard deviation (SD) are calculated. May be. The amount of luminescence emitted from the cells to which the blood sample of the subject was added is the average value of the amounts of luminescence (integrated values) emitted from the cells to which the blood sample of the normal human population was added + nSD (for example, n = 1, 2, 3, 4 Alternatively, if it is higher than 5), it can be judged that the concentration of the TSHR-stimulating antibody in the blood of the subject is higher than that of a normal person, and the subject can be diagnosed as having Graves' disease.
In the present specification and claims, the value obtained from “a sample derived from normal human blood” refers to a measured value of a normal human blood sample (standard product) or a measured value of a normal human population unless otherwise specified. It may be a value obtained in advance.

Alternatively, the light emission amount (integrated value) emitted from the cells to which the blood sample of the subject was added / the light emission amount (integrated value) X100 (%) emitted from the cells to which the blood sample of a normal person was added may be calculated (calculation). Value is calculated value A). The amount of luminescence (integrated value) emitted from a large number of normal human populations and a large number of untreated Graves 'disease patient populations, for example, cells each containing 50 to 100 blood samples, is measured to determine the presence of Graves' disease. Cut-offs in which the disease diagnosis rate and / or the disease-free accuracy rate of being a normal person are, for example, 80% or more, 90% or more, 92% or more, 94% or more, 96% or more, or 98% or more, respectively A value may be set. If the calculated value A is higher than the cut-off value, the subject can be diagnosed with Graves' disease. The cut-off value can be appropriately set according to the nature of the target group such as age and sex, but for example, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200% It can be.
Further, a standard curve of an antibody (TSAb) (for example, NIBSC 90/672 or 65/122) may be used to prepare a calibration curve by serially diluting the concentration. With reference to the calibration curve, the blood concentration of the actual antibody (TSAb) is calculated from the amount of luminescence of the calcium sensitive protein emitted from the cells to which the blood sample of the subject was measured, and a number of normal persons measured in advance. By comparing a population and a large number of untreated Graves' disease patient populations, for example, blood concentrations of antibodies (TSAb) of 50-100 populations each, or known data reported in literature, etc. It is also possible to diagnose whether or not. For example, a subject can be diagnosed as having Graves' disease if it is higher than the mean value of the blood concentration of (TSAb) in the normal population + nSD (eg, n = 1, 2, 3, 4 or 5).
Regarding the measurement of the amount of luminescence, it is preferable to measure an integrated value for a fixed time, for example, an integrated value for 5 minutes, 10 seconds, 15 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute is measured. be able to.

In addition, when the action mechanism described in (2) (a) above is used, hypothyroidism (including Hashimoto's disease) can be diagnosed using the composition according to the present invention. For example, the amount of luminescence of calcium-sensitive protein induced by TSH in cells to which a blood sample of a subject is added and TSH in cells to which a normal blood sample (standard product) of the same amount as that of the subject's blood sample is added. Hypothyroidism can be diagnosed by measuring the amount of luminescence of calcium sensitive protein. In this case, if the amount of luminescence emitted from the cells added with the blood sample of the subject is lower than the amount of luminescence emitted from the cells added with the blood sample (standard product) of a normal person, the TSHR-inhibiting antibody in the blood of the subject It can be judged that the concentration of (TSBAb) is higher than that of a normal person, and the subject can be diagnosed as having hypothyroidism.
In addition, the amount of luminescence (integrated value) of calcium-sensitive protein induced by TSH in cells to which a blood sample of a large number of normal people, for example, 50 to 100 normal people was added, was measured, and the average value and A standard deviation (SD) may be calculated. The amount of luminescence induced by TSH in cells to which the blood sample of the subject was added is the average value of the amount of luminescence (integrated value) induced by TSH in the cells to which the blood sample of the normal human population was added −nSD (for example, n = If it is lower than 1, 2, 3, 4 or 5), it can be judged that the concentration of the TSHR-inhibiting antibody (TSBAb) in the blood of the subject is higher than that of a normal person, and the subject is diagnosed with hypothyroidism can do.
Alternatively, the light emission amount (integrated value) emitted from the cells to which the blood sample of the subject was added / the light emission amount (integrated value) X100 (%) emitted from the cells to which the blood sample of a normal person was added may be calculated (calculation). Value is calculated value B). In advance, the amount of luminescence (integrated value) attributed to blood samples of a large number of normal human populations and a large number of untreated hypothyroid patients, for example, 50 to 100 each, is measured to determine whether hypothyroidism is present. Cut-offs in which the disease diagnosis rate and / or the disease-free accuracy rate of being a normal person are, for example, 80% or more, 90% or more, 92% or more, 94% or more, 96% or more, or 98% or more, respectively When the value is set and the calculated value B is lower than the cut-off value, the subject can also be diagnosed with hypothyroidism. The cut-off value can be appropriately set depending on the nature of the target group such as age and sex, but for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% It can be.

Further, a standard curve of an antibody (TSBAb) having a known concentration may be used to prepare a calibration curve by serially diluting the concentration. With reference to the calibration curve, the blood concentration of the actual antibody (TSBAb) was calculated from the amount of luminescence of the calcium-sensitive protein induced by TSH in the cells to which the blood sample of the subject was measured. Normal population and a large number of untreated hypothyroid patients population, for example, blood concentration of antibody (TSBAb) in each population of 50-100 each, or comparison with known data reported in literature etc. Thus, it can be diagnosed whether or not the subject has hypothyroidism. For example, a subject can be diagnosed with hypothyroidism if it is higher than the mean blood concentration of (TSBAb) in the normal population + nSD (eg, n = 1, 2, 3, 4 or 5). .
Regarding the measurement of the amount of luminescence, it is preferable to measure an integrated value for a fixed time, for example, an integrated value for 5 minutes, 10 seconds, 15 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute is measured. be able to.
In addition, when the action mechanism described in (2) and (b) above is used, hypothyroidism (including Hashimoto's disease) can be diagnosed using the composition according to the present invention. For example, the amount of calcium-sensitive protein luminescence caused by forskolin in cells added with the subject's blood sample, and forskolin in cells added with the same amount of normal human blood sample (standard) as the subject's blood sample Hypothyroidism can be diagnosed by measuring the amount of luminescence of the induced calcium sensitive protein. In this case, if the amount of luminescence emitted from the cells to which the blood sample of the subject is added is higher than the amount of luminescence emitted from the cells to which the blood sample (standard product) of a normal person is added, the TSHR-inhibiting antibody in the blood of the subject It can be judged that the concentration of (TSBAb) is higher than that of a normal person, and the subject can be diagnosed as having hypothyroidism.
In addition, the luminescence amount (integrated value) of calcium-sensitive protein induced by forskolin in cells to which a blood sample of a large number of normal people, for example, 50 to 100 normal people was added, was measured in advance, and the average value thereof And standard deviation (SD) may be calculated. The amount of luminescence induced by forskolin in the cells to which the blood sample of the subject was added is the average value of the amount of luminescence (integrated value) induced by forskolin in the cells to which the blood sample of the normal population was added + nSD (for example, nSD = 1, 2, 3, 4 or 5), it can be judged that the concentration of TSHR-inhibiting antibody (TSBAb) in the blood of the subject is higher than that of normal subjects, and the subject has hypothyroidism. It can also be diagnosed.

Alternatively, the amount of luminescence emitted from the cells to which the blood sample of the subject was added (integrated value) / the amount of luminescence to be emitted from the cells to which the blood sample of normal subjects was added (integrated value) X100 (%) was calculated (calculated value was calculated). Value C). In advance, the amount of luminescence (integrated value) attributed to blood samples of a large number of normal human populations and a large number of untreated hypothyroid patients, for example, 50 to 100 each, is measured to determine whether hypothyroidism is present. Cut-offs in which the disease diagnosis rate and / or the disease-free accuracy rate of being a normal person are, for example, 80% or more, 90% or more, 92% or more, 94% or more, 96% or more, or 98% or more, respectively If the value is set and the calculated value C is higher than the cut-off value, the subject can also be diagnosed with hypothyroidism. The cut-off value can be appropriately set depending on the properties of the target group such as age and sex, but for example, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800% or 900% It can be.
Further, a standard curve of an antibody (TSBAb) having a known concentration may be used to prepare a calibration curve by serially diluting the concentration. The blood concentration of the actual antibody (TSBAb) was calculated from the amount of luminescence of calcium-sensitive protein induced by forskolin in the cells to which the blood sample of the measured subject was added, and many normal human populations and many By comparing the untreated hypothyroid patient population, for example, the blood concentration of the antibody (TSBAb) of each population of 50 to 100 each, or known data reported in the literature, the subject is hypothyroid It is also possible to diagnose whether or not the patient is ill. For example, a subject can be diagnosed with hypothyroidism if it is higher than the mean blood concentration of (TSBAb) in the normal population + nSD (eg, n = 1, 2, 3, 4 or 5). .
Regarding the measurement of the amount of luminescence, it is preferable to measure an integrated value for a fixed time, for example, an integrated value for 5 minutes, 10 seconds, 15 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute is measured. be able to.

In addition, by using the composition according to the present invention, a human who has a high risk of developing a thyroid disease, for example, a human who has a high risk of developing Graves' disease or hypothyroidism can be determined.
For example, if the concentration of a TSHR-stimulating antibody in the blood using the composition according to the present invention is lower than that of a Graves 'disease patient and higher than that of a normal person at the time of a health check, Graves' disease is Risk of developing hypothyroidism if it can be determined that humans are at high risk of developing and the concentration of TSHR binding-inhibiting antibodies in the blood is lower than that of hypothyroidism and higher than that of normal individuals It can be judged as a highly human. In addition, if the concentration of TSHR-stimulating antibody in the blood is gradually increased over time using the composition according to the present invention at the time of a health checkup, a human who has a high risk of developing Graves' disease If the concentration of the TSHR binding inhibitory antibody in the blood gradually increases over time, it can be determined that the human is at high risk of developing hypothyroidism.

Furthermore, the effectiveness of the treatment of humans who have been treated for thyroid diseases, for example, those who have developed Graves' disease or hypothyroidism by using the composition according to the present invention, and are undergoing such treatment. Can be judged.
For example, using the composition according to the present invention, the concentration of TSHR-stimulating antibody or TSHR binding-inhibiting antibody in blood samples collected from the same individual both before and after treatment, The presence or absence of treatment effectiveness can be determined by measuring, and if the concentration of TSHR-stimulating antibody in the blood is reduced after treatment compared to before treatment using the composition according to the present invention, Graves' disease If the concentration of the TSHR binding-inhibiting antibody in the blood is reduced before treatment compared to after treatment, it can be judged that the treatment for hypothyroidism was effective.

In addition to the composition containing cells expressing the thyroid stimulating hormone receptor (TSHR), cAMP-dependent calcium channel, and calcium-sensitive protein according to the present invention, the present invention includes a cell culture solution, a detection solution, and luminescence of the calcium-sensitive protein. Provided is a kit that can contain at least one selected from the group consisting of a substrate or an aqueous solution thereof, an antibody separation solution, a cell culture plate or test tube, TSH or an aqueous solution thereof, an anti-TSH antibody, and a normal human IgG control serum. For example, the kit can be prepared by individually packing the substances and compositions constituting the kit and putting them together in a container such as a box.
Cells expressing the thyroid stimulating hormone receptor (TSHR), cAMP-dependent calcium channel, and calcium-sensitive protein according to the present invention can be prepared as appropriate, for example, at a concentration of 3 × 10 ⁴ cells / ml to 3 × 10 ⁶ cells / ml. Is done. The cells according to the present invention can be prepared by suspending in an aqueous solution at a concentration of 3 × 10 ⁶ cells / ml, for example. When a 96-well plate is used as the cell culture plate, the cells can be seeded at, for example, ³ × 10 ³ cells to ³ × 10 ⁵ cells per well of the 96-well plate.
The cell culture medium is not limited as long as it can maintain the cells according to the present invention, and may be Ca ²⁺ free or Ca ^2+, Mg ²⁺ free.
The detection solution may contain CaCl ₂ , trypan blue, a cation capable of replacing calcium capable of emitting aequorin (for example, cadmium ion, strontium ion), magnesium ion, zinc ion, sulfate ion and / or carbonate ion. The concentration of CaCl ₂ and trypan blue can be appropriately set by those skilled in the art. For example, the concentration of CaCl ₂ is 9 to 18 mM, and the concentration of trypan blue is 0.001 to 0.010%. For example, the detection solution is an aqueous solution containing 9 mM CaCl ₂ and 0.002% trypan blue. The final concentration of CaCl ₂ when measuring the amount of luminescence of the calcium sensitive protein can be 3 to 6 mM. Further, for example, by dissolving a cation, magnesium ion, zinc ion, sulfate ion and / or carbonate ion that can be replaced with calcium in the detection solution, the detection solution can be replaced with calcium, cation, magnesium ion, or zinc ion. , Sulfate ions and / or carbonate ions.
The luminescent substrate of the calcium sensitive protein includes coelenterazine or a coelenterazine derivative that is a luminescent substrate of aequorin, and the derivative of coelenterazine includes ViviRen (registered trademark, Promega). The concentration of ViviRen can be set as appropriate, and is, for example, 0.6 to 30 mM. For example, an aqueous solution of a calcium-sensitive protein luminescent substrate is a 4 mM ViviRen (Promega) aqueous solution. The final concentration of ViviRen when measuring the amount of luminescence of the calcium sensitive protein can be 0.24 to 12 μM.
The antibody separation solution only needs to recover a TSHR-stimulating antibody or a TSHR binding-inhibiting antibody from a blood sample, and can contain 10 to 30% of PEG6000. For example, the antibody separation solution may be an aqueous solution containing 30% PEG 6000.
An example of a cell culture plate is a 96-well plate that can measure the amount of luminescence with a luminometer and can culture cells.
The test tube is not particularly limited and can be appropriately selected by those skilled in the art. For example, a test tube suitable for an apparatus for measuring luminescence emitted from a calcium sensitive protein can be used.
TSH should just be used as an agonist of TSHR, and can be used as a positive control. The TSH can be, but is not limited to, bovine-derived TSH. TSH can be appropriately prepared by those skilled in the art and can be adjusted to 0.01 to 100 mU / ml. For example, bovine-derived TSH is prepared as a 1 mU / ml aqueous solution. The final concentration of bovine-derived TSH when measuring the luminescence of calcium-sensitive protein can be 0.6 μU / ml to 6 mU / ml. For example, the final concentration of bovine-derived TSH when measuring the amount of luminescence of calcium-sensitive protein is 100 μU / ml. Also, TSAb may be used instead of or in addition to TSH. The TSAb can be a monoclonal antibody or a polyclonal antibody.
Normal human IgG control serum can be used as a negative control and can be appropriately prepared by those skilled in the art.

The anti-TSH antibody may be a polyclonal antibody or a monoclonal antibody. The anti-TSH antibody can be an antibody derived from a suitable mammal, and includes mouse anti-TSH antibody, rat anti-TSH antibody, rabbit anti-TSH antibody, goat anti-TSH antibody. The anti-TSH antibody can be appropriately modified by those skilled in the art. In addition, TSH as an antigen is appropriately selected, and TSH as an antigen includes human TSH, mouse TSH, rat TSH, rabbit TSH, cat TSH, and dog TSH. Examples of the anti-TSH antibody used in the kit of the present invention include goat anti-human TSH polyclonal antibody. A person skilled in the art can appropriately set the concentration of the anti-TSH antibody. For example, the concentration of the anti-TSH antibody solution packed in the kit may be 0.01 μg / ml to 100 μg / ml. The final concentration of the anti-TSH monoclonal antibody when measuring the luminescence of the calcium sensitive protein can be, for example, 0.05 to 5.48 μg / ml.
Among patients with hypothyroidism, there are patients with high levels of TSH in the blood. In this case, since calcium-sensitive protein emits light in cells by TSH, Graves' disease can be diagnosed despite being a patient with hypothyroidism. However, by adding a blood sample derived from a patient with hypothyroidism having a high TSH amount to the cells according to the present invention together with an anti-TSH antibody, the patient-derived TSH is neutralized. Accidental diagnosis of disease can be avoided. In addition, when an anti-TSH antibody is added to a cell of the present invention together with a blood sample derived from a patient, and from a calcium sensitive protein when a blood sample derived from a patient is added to a cell of the present invention without adding an anti-TSH antibody. By comparing the amount of emitted light, information on the amount of TSH in the blood of the patient can be obtained. By combining this information with the patient's clinical symptoms, doctors can more accurately diagnose thyroid diseases.

The kit of the present invention further includes serum of a patient with thyroid disease, for example, serum of Graves' disease patient and / or hypothyroidism patient, and these sera are used for diagnosis of thyroid disease and risk of developing the disease. It can be used as a control or a standard for concentration calculation in determination and determination of the effectiveness of treatment of the disease.

  Furthermore, the kit of the present invention may contain a substance that activates adenylate cyclase, such as forskolin. A sample derived from the blood of a subject is added to cells together with TSH, and after culturing for a certain period of time, the presence or absence of a TSHR binding inhibitory antibody in the sample can be determined by adding forskolin. It is also possible to measure the concentration of TSHR binding-inhibiting antibody in the sample, thereby diagnosing hypothyroidism (eg, Hashimoto's disease) and developing hypothyroidism (eg, Hashimoto's disease) Human beings who are at risk of being treated, and the therapeutic effect of patients who have been treated for hypothyroidism (eg, Hashimoto's disease) can be determined.

  The composition or kit according to the present invention is also useful as a diagnostic aid for doctors to diagnose Graves' disease and / or hypothyroidism, taking into account the patient's clinical symptoms and / or other test results. is there. For example, measuring the amount of TSHR-stimulating antibody (TSAb) in a blood sample of a patient exhibiting hyperthyroidism using the composition or kit according to the present invention is based on Graves' disease and destructive hyperthyroidism ( For example, it may be useful for differential diagnosis from painless thyroiditis, subacute thyroiditis).

The present invention also includes the following steps (A) to (C):
(A) Thyroid-stimulating hormone receptor (TSHR), cAMP-dependent calcium channel, and cells expressing calcium-sensitive protein, calcium-sensitive protein luminescent substrate, Ca ²⁺ -free medium, and samples from subject's blood Preparing a mixture;
(B) adding a Ca ²⁺ containing solution to the mixture prepared in (A); and (C) measuring the luminescence of calcium sensitive protein generated from the cells.
And a method for determining Graves' disease.
A medium containing no Ca ²⁺ used in step (A) of the above method can be appropriately selected by those skilled in the art, and may be a medium containing no Ca ²⁺ or Mg ²⁺ .
The Ca ²⁺ containing solution used in step (B) of the above method can be appropriately selected by those skilled in the art, and may be, for example, a CaCl ₂ solution. The Ca ²⁺ -containing solution may further contain a cation capable of replacing calcium (for example, cadmium ion, strontium ion), magnesium ion, zinc ion, sulfate ion and / or carbonate ion.
The mixture prepared in step (A) of the above method may further contain an anti-TSH antibody. By including the anti-TSH antibody in the mixture prepared in step (A) of the above method, it can be avoided to erroneously determine Graves' disease in patients with hypothyroidism in which the amount of TSH in the blood is high.
The order in which the substances described in step (A) of the above method are added can be appropriately determined by those skilled in the art.
For example, the present invention includes the following steps:
(1) culturing cells expressing thyroid stimulating hormone receptor (TSHR), cAMP-dependent calcium channel, and calcium-sensitive protein in a Ca ²⁺ -free medium supplemented with a luminescent substrate of calcium-sensitive protein;
(2) adding a sample derived from the blood of the subject to the cultured cells and further culturing; and (3) adding a CaCl ₂ solution to the cultured cells and measuring the luminescence of the calcium sensitive protein generated from the cells. A method for determining Graves' disease is provided.
In this method, the cells may be stored frozen, in which case they can be thawed by a gentle operation such as a warm bath. The thawed cells may be cultured in a medium containing no Ca ²⁺ or Mg ²⁺ supplemented with a luminescent substrate of a calcium sensitive protein. Luminescent substrates for calcium sensitive proteins include coelenterazine and ViviRen®.
The Ca ²⁺ -free medium may be any medium that can maintain cells. For example, 130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl ₂ , 4.8 mM NaHCO ₃ , 5% PEG6000, pH 7.4 is used. In addition, the Ca ²⁺ -free medium can be a Ca ²⁺ and Mg ²⁺ -free medium. The cells may be seeded in an appropriate container at an appropriate concentration. For example, the cells are seeded at 90 μl / well at 3 to 30 × 10 ⁴ cells / ml in a 96-well plate capable of accommodating a luminometer. The culture time in the step (1) may be 2 hours or more, and can be set at 2-8 hours, for example, 3 hours. In general, in order to recover from damage caused by passage and increase the amount of luminescence of calcium-sensitive proteins such as aequorin, the culture is usually carried out for about 12-24 hours after cell seeding. Even when cultured for about 2 hours, cell death due to the additive was not induced, and it was confirmed that the calcium sensitive protein showed strong luminescence. For example, using aequorin as a calcium-sensitive protein, optimizing the codon of its DNA sequence to human type, adding a mitochondrial translocation signal, and further adopting ViviRen (registered trademark) as a luminescent substrate for calcium-sensitive protein Thus, it was confirmed that strong luminescence from the calcium-sensitive protein can be detected even for about 2 hours after cell seeding.
The blood-derived sample added in the step (2) is prepared by adding an aqueous PEG solution to the blood of a subject or the like and collecting the precipitated fraction. As an example of an aqueous PEG solution, 30% PEG6000 is used. In some cases, a sample derived from the blood of a Graves' disease patient can be used as a positive control, and / or a blood sample derived from a normal person can be used as a negative control, and added to each cell in step (2). The culture time in the step (2) is not limited, but can be 30 to 60 minutes, for example, 30 minutes. Thereby, compared with the case where cAMP is accumulated in the cells and there is no culture after the addition of the sample, more stable and strong luminescence can be measured immediately after the addition of the CaCl ₂ solution.
If the total culture time of step (2) and step (3) is within about 4 hours, the time is short and it is not necessary to be aseptic culture.
In step (2), an anti-TSH antibody may be further added to the cultured cells. The anti-TSH antibody may be a polyclonal antibody or a monoclonal antibody. The anti-TSH antibody can be an antibody derived from a suitable mammal, and includes mouse anti-TSH antibody, rat anti-TSH antibody, rabbit anti-TSH antibody, goat anti-TSH antibody. Those skilled in the art can appropriately modify the anti-TSH antibody. In addition, TSH as an antigen is appropriately selected, and TSH as an antigen includes human TSH, mouse TSH, rat TSH, rabbit TSH, cat TSH, and dog TSH. Examples of the anti-TSH antibody used in the method of the present invention include goat anti-human TSH polyclonal antibody.
The CaCl ₂ -containing solution used in step (3) may further contain a cation (for example, cadmium ion, strontium ion), magnesium ion, zinc ion, sulfate ion and / or carbonate ion that can be substituted for calcium. The concentration of Ca ²⁺ , cation capable of replacing calcium, magnesium ion, zinc ion, sulfate ion and carbonate ion that can be contained in CaCl ₂ solution can maintain the cell, and calcium sensitive proteins such as aequorin are adequate It can be set appropriately by those skilled in the art to emit light.
In step (3), the luminescence of calcium-sensitive proteins such as aequorin is performed immediately after the addition of the CaCl ₂ solution, and can be measured by methods well known by those skilled in the art. For example, using a luminometer (PerkinElmer, ARVO-Sx) capable of continuously stirring and measuring automatically, the amount of luminescence can be measured by integrating the luminescence values for 15-30 seconds after stirring. A person skilled in the art can appropriately select an apparatus that can be used for measuring the amount of luminescence.

  As a result of carrying out the above method, the amount of luminescence of calcium-sensitive proteins such as aequorin generated from cells to which the blood-derived sample of the subject is added is calcium-sensitive such as aequorin that is generated from cells to which the normal blood sample is added. If it is higher than the amount of luminescence of the protein, it can be determined as Graves' disease. Further, by determining the antibody concentration in blood and comparing it with the standard antibody concentration of a Graves 'disease patient and / or a normal person, it can be determined whether or not it is Graves' disease.

For example, the amount of luminescence emitted from cells to which a subject's blood sample was added and the amount of luminescence emitted from cells to which a normal human blood sample (standard product) was added in the same amount as the subject's blood sample were measured. If the amount of luminescence emitted from the cell to which the sample is added is higher than the amount of luminescence emitted from the cell to which a normal human blood sample (standard product) is added, the concentration of the TSHR stimulating antibody (TSAb) in the blood of the subject is increased. It can be determined that it is higher than that of normal persons, and the subject can be determined to have Graves' disease.
In addition, the amount of luminescence (integrated value) when a blood sample of a large number of normal people, for example, 50 to 100 normal people is added, is measured in advance, and the average value and standard deviation (SD) are calculated. Also good. The amount of luminescence emitted from the cells to which the blood sample of the subject was added is the average value of the amounts of luminescence (integrated values) emitted from the cells to which the blood sample of the normal human population was added + nSD (for example, n = 1, 2, 3, 4 Alternatively, if it is higher than 5), it can be determined that the concentration of the TSHR-stimulating antibody in the blood of the subject is higher than that of a normal person, and the subject can be determined to have Graves' disease.

Further, the amount of luminescence emitted from the cells added with the blood sample of the subject (integrated value) / the amount of luminescence emitted from the cells added with the blood sample of normal subjects (integrated value) X100 (%) is calculated (calculated value is calculated). Value D). The amount of luminescence (integrated value) emitted from a large number of normal human populations and a large number of untreated Graves 'disease patient populations, for example, cells each containing 50 to 100 blood samples, is measured to determine the presence of Graves' disease. Cut-offs in which the disease diagnosis rate and / or the disease-free accuracy rate of being a normal person are, for example, 80% or more, 90% or more, 92% or more, 94% or more, 96% or more, or 98% or more, respectively When the value is set and the calculated value D is higher than the cutoff value, the subject can also be determined to have Graves' disease. The cut-off value can be appropriately set according to the nature of the target group such as age and sex, but for example, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200% It can be.
Furthermore, a standard curve of an antibody (TSAb) (for example, NIBSC 90/672 or 65/122) may be used to prepare a calibration curve by serially diluting the concentration. With reference to the calibration curve, the blood concentration of the actual antibody (TSAb) was calculated from the luminescence amount of the calcium sensitive protein in the cells to which the blood sample of the subject was measured, and a large number of normal human populations measured in advance and The subject has Graves 'disease by comparing the blood concentration of antibody (TSAb) of a large number of untreated Graves' disease patients, for example, 50-100 each, or known data reported in literature, etc. It can also be determined whether or not. For example, the subject can also be determined to have Graves' disease if it is higher than the mean blood concentration of the normal population (TSAb) + nSD (eg, n = 1, 2, 3, 4 or 5).

In addition, as a result of carrying out the above method, the amount of luminescence of calcium-sensitive proteins such as aequorin generated from cells to which the blood-derived sample of the subject was added is such as aequorin generated from cells to which the normal blood sample was added. The risk of developing Graves 'disease if it is high compared to the amount of luminescence of calcium-sensitive protein and low compared to the amount of luminescence of calcium-sensitive proteins such as aequorin generated from cells to which a blood-derived sample from a patient with Graves' disease is added. It can be determined that the person is a high person. Alternatively, it is possible to determine whether or not a person is at high risk of developing Graves 'disease by determining the antibody concentration in the blood and comparing it with the standard antibody concentration of Graves' disease patients and / or normal persons. it can.
Further, in step (2), before and after treatment of the same Graves' disease patient as a sample derived from the subject's blood, the amount of luminescence of calcium sensitive proteins such as aequorin generated from the cells is compared. Thus, the effectiveness of the treatment can be determined. As a result of the test, if the luminescent amount of calcium sensitive protein such as aequorin is lower in the sample after treatment than in the sample before treatment, it can be determined that the treatment was effective.

The present invention also includes the following steps (A) to (C):
(A) Thyroid-stimulating hormone receptor (TSHR), cAMP-dependent calcium channel, and cells expressing calcium-sensitive protein, calcium-sensitive protein luminescent substrate, Ca ²⁺ -free medium, TSH or stimulated TSAb monoclonal antibody, And preparing a mixture comprising a sample from the subject's blood;
(B) adding a Ca ²⁺ containing solution to the mixture prepared in (A); and (C) quantifying the luminescence of the calcium sensitive protein generated from the cells.
And a method for determining hypothyroidism.
A medium containing no Ca ²⁺ used in step (A) of the above method can be appropriately selected by those skilled in the art, and may be a medium containing no Ca ²⁺ or Mg ²⁺ .
The Ca ²⁺ containing solution used in step (B) of the above method can be appropriately selected by those skilled in the art, and may be, for example, a CaCl ₂ solution. The Ca ²⁺ -containing solution may further contain a cation (for example, cadmium ion, strontium ion), magnesium ion, zinc ion, sulfate ion and / or carbonate ion that can be substituted for calcium.
The order in which the substances described in step (A) of the above method are added can be appropriately determined by those skilled in the art.
For example, the present invention includes the following steps:
(1) culturing cells expressing thyroid stimulating hormone receptor (TSHR), cAMP-dependent calcium channel, and calcium-sensitive protein in a Ca ²⁺ -free medium supplemented with a luminescent substrate of calcium-sensitive protein;
(2) A step of adding a sample derived from a subject's blood to cultured cells together with TSH and further culturing; and (3) adding a CaCl ₂ solution to the cultured cells and measuring luminescence of calcium sensitive protein generated from the cells. A method for determining hypothyroidism (for example, Hashimoto's disease) is provided.
Steps (1) to (3) can be appropriately performed by those skilled in the art with reference to the method for determining Graves' disease.
Optionally, in step (2), a sample from normal human blood can be used as a negative control and / or a sample from the blood of a hypothyroid patient as a positive control, which can be added to each cell. Moreover, although the culture | cultivation time in a process (2) is not limited, It can be set to 30-120 minutes, for example, can be set to 30 minutes. Further, in step (2), the TSH can be bovine-derived TSH, and optionally a TSAb can be added instead of or in addition to TSH. The TSAb can be a monoclonal antibody.

As a result of carrying out the above method, the amount of luminescence of calcium-sensitive proteins such as aequorin generated from cells to which the blood-derived sample of the subject was added is calcium-sensitive such as aequorin generated from cells to which the normal blood sample was added. If it is lower than the amount of luminescence of the protein, it can be determined as hypothyroidism. Further, by determining the antibody concentration in the blood, it is possible to determine whether or not the patient has hypothyroidism by comparing with the standard antibody concentration of hypothyroid patients and / or normal persons.
For example, the amount of luminescence emitted from cells to which a subject's blood sample was added and the amount of luminescence emitted from cells to which a normal human blood sample (standard product) was added in the same amount as the subject's blood sample were measured. If the amount of luminescence emitted from the cell to which the sample is added is lower than the amount of luminescence emitted from the cell to which a normal human blood sample (standard product) is added, the concentration of the TSHR-inhibiting antibody (TSBAb) in the blood of the subject is It can be determined that it is higher than that of a normal person, and the subject can be determined to have hypothyroidism.
In addition, the amount of luminescence (integrated value) of calcium-sensitive protein by TSH in cells to which a blood sample of a large number of normal people, for example, 50 to 100 normal people was added, was measured in advance, and the average value and standard deviation ( SD) may be calculated. The amount of luminescence by TSH in the cells to which the blood sample of the subject was added is the average value of the amount of luminescence (integrated value) of TSH in the cells to which the blood sample of the normal human population was added-nSD (for example, n = 1, 2, 3, If it is lower than 4 or 5), it can be judged that the concentration of the TSHR-inhibiting antibody (TSBAb) in the blood of the subject is higher than that of a normal person, and the subject can also be judged to have hypothyroidism.

Further, the amount of luminescence when the blood sample of the subject is added (integrated value) / the amount of luminescence when the blood sample of the normal person is added (integrated value) X100 (%) is calculated (the calculated value is set as the calculated value E). ) In advance, the amount of luminescence (integrated value) attributed to blood samples of a large number of normal human populations and a large number of untreated hypothyroid patients, for example, 50 to 100 each, is measured to determine whether hypothyroidism is present. Cut-offs in which the disease diagnosis rate and / or the disease-free accuracy rate of being a normal person are, for example, 80% or more, 90% or more, 92% or more, 94% or more, 96% or more, or 98% or more, respectively If the value is set and the calculated value E is lower than the cut-off value, the subject can also be determined to have hypothyroidism. The cut-off value can be appropriately set depending on the nature of the target group such as age and sex, but for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% It can be.
Further, a standard curve of an antibody (TSBAb) having a known concentration may be used to prepare a calibration curve by serially diluting the concentration. With reference to the calibration curve, the blood concentration of the actual antibody (TSBAb) is calculated from the amount of luminescence emitted from the cells to which the blood sample of the subject was measured, and a large number of normal human populations and many By comparing the untreated hypothyroid patient population, for example, the blood concentration of the antibody (TSBAb) of each population of 50 to 100 each, or known data reported in the literature, the subject is hypothyroid It can also be determined whether or not the patient is ill. For example, a subject can also be determined to have hypothyroidism if it is higher than the mean blood concentration of (TSBAb) in the normal population + nSD (eg, n = 1, 2, 3, 4 or 5). .
In addition, as a result of carrying out the above method, the amount of luminescence of calcium-sensitive proteins such as aequorin generated from cells to which the blood-derived sample of the subject was added is such as aequorin generated from cells to which the normal blood sample was added. If it is low compared to the amount of luminescence of calcium-sensitive protein and high compared to the amount of luminescence of aequorin generated from cells to which a blood-derived sample from a hypothyroid patient is added, there is a risk of developing hypothyroidism. It can be determined that the person is high. Alternatively, by determining the antibody concentration in the blood and comparing it with the standard antibody concentration of hypothyroid patients and / or normal individuals, it is determined whether or not the person is at high risk of developing hypothyroidism. Can be determined.
Furthermore, in step (2), before and after treatment of the same hypothyroid patient as a sample derived from the blood of the subject, the amount of luminescence of calcium sensitive protein such as aequorin generated from the cells Can be used to determine the effectiveness of the treatment. As a result of the test, if the sample after treatment has a higher luminescence amount of calcium sensitive proteins such as aequorin than before treatment, it can be determined that the treatment was effective.

The present invention further includes the following steps (A) to (C):
(A) A mixture comprising the cell according to any one of claims 1 to 10, a luminescent substrate of a calcium sensitive protein, a Ca ²⁺ -containing medium, a TSH or stimulated TSAb monoclonal antibody, and a sample derived from blood of a subject is prepared. Do;
(B) adding forskolin to the mixture prepared in (A); and
(C) quantifying the luminescence of the calcium sensitive protein generated from the cells,
And a method for determining hypothyroidism.
A Ca ²⁺ -containing medium used in step (A) of the above method can be appropriately selected by those skilled in the art. For example, it may be a medium containing CaCl ₂ . The Ca ²⁺ -containing medium may further contain a cation (for example, cadmium ion, strontium ion), magnesium ion, zinc ion, sulfate ion and / or carbonate ion that can be substituted for calcium. Concentrations of Ca ²⁺ , cations that can replace calcium, magnesium ions, zinc ions, sulfate ions and carbonate ions that can be contained in a Ca ²⁺ -containing medium can maintain the cells, and calcium sensitive proteins such as aequorin It can be appropriately set by those skilled in the art to appropriately emit light.
The order in which the substances described in step (A) of the above method are added can be appropriately determined by those skilled in the art.
For example, the present invention includes the following steps:
(1) culturing cells expressing a thyroid stimulating hormone receptor (TSHR), a cAMP-dependent calcium channel, and a calcium sensitive protein in a Ca ²⁺ -containing medium supplemented with a luminescent substrate of the calcium sensitive protein;
(2) adding a sample derived from the blood of a subject to cultured cells together with TSH and further culturing; and
(3) Provided is a method for determining hypothyroidism (for example, Hashimoto's disease), comprising the step of adding forskolin to cultured cells and measuring the luminescence of calcium-sensitive protein generated from the cells.
Steps (1) to (3) can be appropriately performed by those skilled in the art with reference to the method for determining Graves' disease.
Optionally, in step (2), a sample from normal human blood can be used as a negative control and / or a sample from the blood of a hypothyroid patient as a positive control, which can be added to each cell. Moreover, although the culture | cultivation time in a process (2) is not limited, It can be 10-120 minutes, for example, can be 10 minutes. Further, in step (2), the TSH can be bovine-derived TSH, and optionally a TSAb can be added instead of or in addition to TSH. The TSAb can be a monoclonal antibody.
In step (3), the concentration of forskolin can be appropriately set by those skilled in the art.

As a result of carrying out the above method, the amount of luminescence of calcium-sensitive proteins such as aequorin generated from cells to which the blood-derived sample of the subject is added is calcium-sensitive such as aequorin that is generated from cells to which the normal blood sample is added. If it is higher than the amount of protein luminescence, it can be determined as hypothyroidism. Further, by determining the antibody concentration in the blood, it is possible to determine whether or not the patient has hypothyroidism by comparing with the standard antibody concentration of hypothyroid patients and / or normal persons.
For example, the amount of luminescence emitted from cells to which a subject's blood sample was added and the amount of luminescence emitted from cells to which a normal human blood sample (standard product) was added in the same amount as the subject's blood sample were measured. If the amount of luminescence emitted from the cell to which the sample is added is higher than the amount of luminescence emitted from the cell to which a normal human blood sample (standard product) is added, the concentration of the TSHR-inhibiting antibody (TSBAb) in the blood of the subject is increased. It can be determined that it is higher than that of a normal person, and the subject can be determined to have hypothyroidism.
In addition, the amount of luminescence (integrated value) when a blood sample of a large number of normal people, for example, 50 to 100 normal people is added, is measured in advance, and the average value and standard deviation (SD) are calculated. Also good. The amount of luminescence emitted from the cells to which the blood sample of the subject was added is the average value of the amounts of luminescence (integrated values) emitted from the cells to which the blood sample of the normal human population was added + nSD (for example, n = 1, 2, 3, 4 Alternatively, if it is higher than 5), it can be determined that the concentration of the TSHR-inhibiting antibody (TSBAb) in the blood of the subject is higher than that of a normal person, and the subject can also be determined to have hypothyroidism.

Further, the amount of luminescence emitted from the cells added with the blood sample of the subject (integrated value) / the amount of luminescence emitted from the cells added with the blood sample of normal subjects (integrated value) X100 (%) is calculated (calculated value is calculated). Value F). In advance, the amount of luminescence (integrated value) attributed to blood samples of a large number of normal human populations and a large number of untreated hypothyroid patients, for example, 50 to 100 each, is measured to determine whether hypothyroidism is present. Cut-offs in which the disease diagnosis rate and / or the disease-free accuracy rate of being a normal person are, for example, 80% or more, 90% or more, 92% or more, 94% or more, 96% or more, or 98% or more, respectively When the value is set and the calculated value F is higher than the cut-off value, the subject can also be determined to have hypothyroidism. The cut-off value can be appropriately set depending on the properties of the target group such as age and sex, but for example, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800% or 900% It can be.
Further, a standard curve of an antibody (TSBAb) having a known concentration may be used to prepare a calibration curve by serially diluting the concentration. With reference to the calibration curve, the blood concentration of the actual antibody (TSBAb) is calculated from the amount of luminescence emitted from the cells to which the blood sample of the subject was measured, and a large number of normal human populations and many By comparing the untreated hypothyroid patient population, for example, the blood concentration of the antibody (TSBAb) of each population of 50 to 100 each, or known data reported in the literature, the subject is hypothyroid It can also be determined whether or not the patient is ill. For example, a subject can also be determined to have hypothyroidism if it is higher than the mean blood concentration of (TSBAb) in the normal population + nSD (eg, n = 1, 2, 3, 4 or 5). .

In addition, as a result of carrying out the above method, the amount of luminescence of calcium sensitive proteins such as aequorin generated from cells to which the blood-derived sample of the subject was added is such as aequorin generated from cells to which the normal blood sample was added. If the amount of luminescence of calcium-sensitive protein is high compared to the amount of luminescence of calcium-sensitive proteins such as aequorin, which is higher than the amount of luminescence of calcium-sensitive protein and is generated from cells to which a sample derived from the blood of a hypothyroid patient is added, hypothyroidism It can be determined that the person has a high risk of developing the disease. Alternatively, by determining the antibody concentration in the blood and comparing it with the standard antibody concentration of hypothyroid patients and / or normal individuals, it is determined whether or not the person is at high risk of developing hypothyroidism. Can be determined.
Furthermore, in step (2), before and after treatment of the same hypothyroid patient as a sample derived from the blood of the subject, the amount of luminescence of calcium sensitive protein such as aequorin generated from the cells Can be used to determine the effectiveness of the treatment. As a result of the test, if the luminescent amount of calcium sensitive protein such as aequorin is lower in the sample after treatment than in the sample before treatment, it can be determined that the treatment was effective.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these.

1. Details of Construction Method of Frozen Cells The cDNA sequence of human thyroid stimulating hormone receptor (Genbank No. NM — 000369) (SEQ ID NO: 5) was amplified by PCR from a cDNA library derived from human thyroid gland and cloned into pUC18. The hTSHR cDNA cloned into pUC18 was excised with BamH1 and recloned into the pZeoSV2 vector (Invitrogen) to create pZeoSV2 hTSHR.
Cyclic nucleotide-dependent calcium channel (Genbank No. BC048775) (SEQ ID NO: 4) from a cDNA library derived from mouse odor epithelial cells was amplified by PCR (1994 bp) and cloned into an expression vector (pCMVSPORT, Invitrogen) and pmCNGα2 (FIG. 18) was created. Furthermore, in order to increase the selectivity and sensitivity to cAMP, a modified cyclic nucleotide in which the 460th cysteine (C) is substituted with tryptophan (W) and the 583th glutamic acid (E) with methionine (M) is substituted with an amino acid. A construct pmCNGα2MW expressing a dependent calcium channel (SEQ ID NO: 6) was prepared by a point mutation PCR method. In addition, a synthetic apoaequorin cDNA sequence (676 bp) (SEQ ID NO: 7) having a mitochondrial translocation signal is optimized with KpnI and NheI, and treated with KpnI and NheI. The obtained DNA was cloned into pcDNA3.1 (Invitrogen) to prepare an apoaequorin expression vector pcDNA mt sAEQ (FIG. 19).
The CHO cell line was seeded in a 10 cm ² petri dish at a cell concentration of 1.0 × 10 ⁵ cells / ml. The next day, 1 μg of pZeoSV2 hTSHR, 2 μg of pmCNGα2MW, and 2 μg of pcDNA mt sAEQ per petri dish were introduced using FuGENE6 ^™ (Roche). On the next day, 400 μL of Versen solution (EDTA) was added to the petri dish, and the cells were detached from the petri dish and suspended in DMEM / F12 medium containing 10 mL of 5% cFCS. The obtained suspension was centrifuged at 1000 rpm for 5 minutes, and then the pellet was dissolved in 1 mL cell banker at a concentration of 2-5 × 10 ⁶ cells / mL and stored at −80 ° C.

2. A concentration-dependent curve of bovine TSH (bTSH) using frozen cells and 1 ml of frozen cells (3 × 10 ⁶ cells / ml) prepared with a minimum detection sensitivity of 1 were lysed in a warm bath and 10 mL of sample buffer (130 mM NaCl, 5 mM KCl, 20 mM HEPES) , 1 mM MgCl ₂ , 4.8 mM NaHCO ₃ , 5% PEG6000, pH 7.4), and after centrifugation at 1000 rpm for 5 minutes, add 10 mL of sample buffer to the resulting precipitate and add 7.5 μL 4 mM ViviRen (Promega) And seeded in a 96-well plate at 90 μL / well. After culturing at 37 ° C CO ₂ for 3 hours, add 10 µL of bTSH serially diluted with PBS (n = 6), incubate for 30 minutes, add 3 mM CaCl ₂ solution at 50 µL / well, and add a luminometer (PerkinElmer, ARVO The amount of luminescence was measured with -Sx, hereinafter the same model was used in the examples. FIG. 1 shows a concentration dependency curve of bTSH.

  As shown in FIG. 2 expanded for the low concentration range, the lowest detection sensitivity of bTSH capable of significantly separating the + 3SD value of the blank value was 0.16 μU / mL. In addition, since aequorin uses a luminescent reaction, this method yielded a high blank / signal ratio (S / N ratio) of 100 μU / mL bTSH S / N ratio, approximately 45 times (9000000/200000). .

As shown in Fig. 2, which was expanded for the low concentration range, the minimum detection sensitivity of bTSH that can significantly separate the + 3SD value of the blank value is 0.16μU / mL, and the minimum detection sensitivity of the existing Yamasa Soy Sauce kit is 1μU / mL. It was one order of magnitude more sensitive than mL (Atsuo Nagata, et al .: Hormone and Clinical, 41: 1023, 1993).
In addition, aequorin uses a luminescence reaction, and the blank / signal ratio (S / N ratio) of 100μU / mL bTSH S / N ratio, about 45 times (9000000/200000) of this method is Compared to 100 μU / mL bTSH SN = 7), this was a significant improvement.

3. Examination of reproducibility TSAb standard product derived from human Graves' patient, MRC Research standard B 1966 Long-acting Thyroid Stimulator (NIBSC ・ Nasional Institute for Biological Standards and Control, code 65/122) is diluted with normal human serum and H (1.88 mU LAST / ml) M (1.25 mU LAST / ml) L (0.94 mU LAST / ml) control samples were prepared. To 50 μL of the prepared control serum, 150 μL of 30% PEG6000 was added and stirred, and then allowed to stand at 4 ° C. for 5 minutes. After centrifugation at 4 ° C. and 3000 rpm for 20 minutes, the resulting precipitate was dissolved in 400 μL sample buffer to obtain a sample solution. 1 ml of frozen cells prepared in 1 (3 × 10 ⁶ cells / ml) is dissolved in a warm bath, suspended in 10 mL of sample buffer, centrifuged at 1000 rpm for 5 minutes, 10 mL of sample buffer is added to the resulting precipitate, and 7.5 μL 4 mM ViviRen And seeded in a 96-well plate at 80 μL / well. After culturing at 37 ° C. CO ₂ for 3 hours, 20 μL of the prepared sample was added (n = 2) and incubated for 30 minutes, and then 50 μL of 9 mM CaCl ₂ solution was added, and the amount of luminescence was measured with a luminometer. When 6 LMH samples are purified and measured simultaneously (simultaneous reproducibility), when purified and measured independently on 10 different days (daily reproducibility), LMH samples are purified on the same day for different production lots And when measured (reproducibility between different lots) was compared. In addition to the measurement sample, H (576 TSAb%), M (342 TSAb%), and L (197 TSAb%), which were separately priced by Yamasa Kit, were added to the plate and a calibration curve was drawn. The value was taken as the sample concentration (Yamasa kit equivalent TSAb%) (Yamasa TSAb% = specimen value / normal human serum value x 100 (%)).
In this method, it was confirmed that high reproducibility was obtained because the measurement system was highly sensitive and the S / N ratio was high. In addition, the CV between different lots in this method was found to be highly reproducible at 5-6%. By using lined CHO cells and optimized gene transfer conditions, a measurement system with little difference between different lots could be constructed.
The reproducibility CV value of the existing Yamasa soy sauce TSAb kit was reported to be 10-15%, confirming the high reproducibility of this method.
In addition, since existing kits are produced by freezing thyroid cells derived from porcine tissue, there is a problem that there is a large variation between lots due to individual differences in pigs from which tissues originate (CV 10 between different lots). However, it was found that this method is highly reproducible even between different lots.

4). Examination of culture time after addition of aequorin luminescent substrate (Viviren) 1 ml of frozen cells prepared in 1 (3 × 10 ⁶ cells / ml) was dissolved in a warm bath, suspended in 10 mL of sample buffer, and obtained after centrifugation at 1000 rpm for 5 minutes. Add 10 mL of sample buffer to the resulting precipitate and add 7.5 μL 4 mM ViviRen. After the addition, 30 minutes, 1, 2, 3, 4, 5, 6, 7, 8 hours, add 90 μL / well to a 96-well plate Sowing. After adding 10 μL of bTSH diluted serially with PBS and culturing for 30 minutes, 9 mM CaCl ₂ solution was added at 50 μL / well, and the luminescence was measured with a luminometer.
It was found that the amount of luminescence reached a plateau 2 hours after Viviren addition (see FIG. 3).

5). Examination of amount of receptor plasmid to be transfected
CHO cells are seeded in 10 cm ² dishes with 10 mL of CHO cells at a concentration of 1 × 10 ⁵ cells / mL, cultured for 1 day, mixed with 3ug pcDNA mt sAEQ, 1ug pmCNGα2MW and 0-1 ug pZeoSV2 TSHR plasmid, and 600 μL DMEM / F12 and 18 μL Fugene6 (Roche) were mixed and transfected. After further overnight culture, the medium was removed, washed with 10 mL PBS, added with 800 μL of versene, incubated at 37 ° C. for 5 minutes, and then suspended in 10 mL of DMEM / F12 cFCS. After centrifuging at 1000 rpm for 5 minutes, dissolve the precipitate in 10 mL of DMEM / F12 cFCS, add 6.5 μL of 4 mM Viviren, incubate at 37 ° C for 3 hours, replace with PBS, and inoculate into a 96-well plate at 90 μL / well did. 30 minutes after adding the bTSH concentration series, 3 mM CaCl ₂ solution was added at 50 μL / well, and the luminescence was measured with a luminometer.
It was revealed that 1 ug / plate amount of transfection was required for the receptor plasmid in order to obtain an optimal amount of luminescence (see FIG. 4).

6). 1 ml of frozen cells (3 × 10 ⁶ cells / ml) prepared in cell concentration optimization 1 are dissolved in a warm bath, suspended in 10 mL of sample buffer, centrifuged at 1000 rpm for 5 minutes, and 10 mL of sample buffer is added to the resulting precipitate. It was. A cell line with a cell concentration adjusted to 3 × 10 ³ cells / mL to 3 × 10 ⁵ cells / mL was prepared, 7.5 μL 4 mM ViviRen was added, and seeded in a 96-well plate at 90 μL / well. After culturing at 37 ° C. CO ₂ for 3 hours, 10 μL of bTSH serially diluted with PBS was added, incubated for 30 minutes, 9 mM CaCl ₂ solution was added at 50 μL / well, and the amount of luminescence was measured with a luminometer.
As shown in FIGS. 5 and 6, the amount of luminescence increased depending on the cell concentration. In terms of relative value with each blank value set to 1, 3 × 10 ⁵ cells / mL was found to be the optimum concentration.

7). Examination of addition concentration of CaCl ₂ detection solution 1 ml of frozen cells (3 × 10 ⁶ cells / ml) prepared in 1 was dissolved in a warm bath, suspended in 10 mL of sample buffer, centrifuged at 1000 rpm for 5 minutes, and 10 mL of the resulting precipitate was added to the resulting precipitate. Sample buffer was added, 7.5 μL 4 mM ViviRen was added, and seeded in a 96-well plate at 90 μL / well. After culturing at 37 ° C. CO ₂ for 3 hours, 10 μL of bTSH serially diluted with PBS was added and incubated for 30 minutes. A CaCl ₂ detection solution having a final concentration of 0 to 30 mM was added at 50 μL / well, and the luminescence was measured with a luminometer.
It was found that a CaCl ₂ concentration of 3 to 6 mM with a low value in the background (0) and a high light emission amount in the sample was optimal (see FIG. 7).

8). Examination of dilution linearity using stimulating antibody control serum 1 ml of frozen cells prepared in 1 (3 × 10 ⁶ cells / ml) were dissolved in a warm bath, suspended in 10 mL of sample buffer, and obtained after centrifugation at 1000 rpm for 5 minutes. To the precipitate, 10 mL of sample buffer was added, 7.5 μL of 4 mM ViviRen was added, and seeded in a 96-well plate at 80 μL / well. The cells were cultured at 37 ° C. CO ₂ for 3 hours. IgG fractions of positive control serum series (Roche NIBSC 90/672 compliant, 40 IU / mL, 13 IU / mL, 8 IU / mL, 4 IU / mL) purified and diluted with 30% PEG 6000 in sample buffer Diluted 1/2, 1/4, 1/8, 1/16, 1/32 times and added 20 μL. After incubation for 30 minutes, 9 mM CaCl ₂ solution was added at 50 μL / well, and the amount of luminescence was measured with a luminometer.
As shown in FIG. 8, it was confirmed that the light emission amount increased with linearity.

9. Examination of detection method of inhibitory antibody (TSBAb) against TSHR 1 ml of frozen cells prepared in 1 (3 × 10 ⁶ cells / ml) were dissolved in a warm bath, suspended in 10 mL of sample buffer, and obtained after centrifugation at 1000 rpm for 5 minutes. To the precipitate, 10 mL of sample buffer was added, 7.5 μL of 4 mM ViviRen was added, and seeded in a 96-well plate at 90 μL / well. After incubation for 3 hours at 37 ° C CO ₂ , add 10 μL of the inhibitory antibody (TSBAb) fraction to TSHR from hypothyroid patients purified with 30% PEG6000 and the IgG fraction from normal human serum, and at the same time bTSH Was added at 10 μL / well to a final concentration of 100 μU to 2.5 μU / mL, incubated for 30 minutes, 9 mM CaCl ₂ solution was added at 50 μL / well, and the luminescence was measured with a luminometer.
In antibody fractions derived from patients with hypothyroidism, it was revealed that 90% or more of luminescence was inhibited in the presence of 2.5 to 10 μU / mL bTSH (see FIG. 9). At 100 μU / mL bTSH, about 50% inhibition was observed, and it was found that competitive inhibition was observed depending on the concentration of bTSH to be competed (see FIG. 9).
When detecting an inhibitory antibody, it is common to see a decrease in signal in the presence of bTSH, but the conventional method measures a low S / N ratio of 5-10 times and a low S / N ratio. Since the disadvantage of narrowing the range was known, this method can detect inhibitory antibodies with high sensitivity because the S / N ratio with and without bTSH is 50 times and the measurement range is wide.

10. Examination of a method for detecting an inhibitory antibody against TSHR (TSBAb) by desensitization of cells 1 ml (3 × 10 ⁶ cells / ml) of frozen cells prepared in 1 was lysed in a warm bath and suspended in 10 ml of sample buffer, and 1000 rpm 5 After centrifugation, DMEM / F12 cFCS medium containing 10 mL of calcium was added to the resulting precipitate, 7.5 μL of 4 mM ViviRen was added, and seeded in a 96-well plate at 90 μL / well. After culturing at 37 ° C. CO ₂ for 3 hours, 10 μL of an inhibitory antibody (TSBAb) fraction against TSHR derived from a hypofunction patient purified with 30% PEG 6000 and an IgG fraction derived from normal human serum were added. At the same time, add bTSH at 10 μL / well to a final concentration of 100 μU to 2.5 μU / mL, incubate for 30 minutes, add 10 ^-4 M Forskoin solution at 50 μL / well, and measure the luminescence with a luminometer. did.
From FIG. 10, at the bTSH concentration of 5 μU or less, desensitization is not sufficient, and there is no difference between the inhibitory antibody and the normal person. However, in the presence of 100 μU bTSH, the luminescence level of normal human serum decreases, An increase in the amount of luminescence was observed in the presence of the inhibitory antibody, and it was found that the presence or absence of the inhibitory antibody could be detected by this method using desensitization.

[Mechanism of luminescence with or without inhibitory antibody in this method]
Under these conditions, when there is no inhibitory antibody such as normal human serum, luminescence reaction occurs immediately after the addition of bTSH because calcium is present in the medium when bTSH is added to the cells. On the other hand, when an inhibitory antibody (TSBAb) is present, TSBAb inhibits the action of bTSH and no luminescence reaction occurs. In this state, when Forskolin that subsequently activates adenylate cyclase is added, cAMP is generated and emits light without passing through the TSH receptor. However, in the absence of TSBAb, the cells are desensitized once by bTSH through the activation of adenylate cyclase, and thus luminescence occurs even when Forskolin, the second stimulus, is further added. do not do. In contrast, when TSBAb is present, bTSH has not activated adenylate cyclase by the inhibitory antibody. Therefore, if Forskolin is added continuously, cAMP is generated and a luminescent reaction occurs.

[Discussion]
The conventional methods for detecting an inhibitory antibody are expressed as a% inhibition rate relative to a normal person. However, when expressed in% inhibition rate, there is a problem that there is no quantitative property in a high concentration range of 90% or more.
In this method using cell desensitization and forskolin, since it can be expressed by the amount of luminescence that increases with respect to normal people, the upper value is not specified, and the presence or absence of an inhibitory antibody is maintained with linearity even in a high concentration range. It can be detected.

11. 1 ml of frozen cells (3 × 10 ⁶ cells / ml) prepared in Sensitivity Comparison with Existing Kit Using Stimulated Antibody Standards were dissolved in a warm bath, suspended in 10 mL sample buffer, and obtained after centrifugation at 1000 rpm for 5 minutes. To the resulting precipitate, 10 mL of sample buffer was added, 7.5 μL of 4 mM ViviRen was added, and seeded in a 96-well plate at 90 μL / well. MRC Research standard B 1966 (NIBSC 65/122) was diluted with normal serum to prepare a concentration series. 150 μL of 30% PEG6000 was added to the prepared 50 μL control series, stirred, and allowed to stand at 4 ° C. for 5 minutes. After centrifugation at 4 ° C. and 3000 rpm for 20 minutes, the resulting precipitate was dissolved in 50 μL sample buffer to obtain a sample solution. After culturing at 37 ° C. CO ₂ for 3 hours, 10 μL of the sample solution was added and incubated for 30 minutes. 9 mM CaCl ₂ detection solution was added at 50 μL / well, and the amount of luminescence was measured with a luminometer.
Separately, the values measured with a kit using an existing porcine thyroid from Yamasa are shown. In the existing Yamasa kit, the concentration is 200 TSAb% at 0.94 mU LAST / mL, and concentrations below that cannot be detected below sensitivity.However, in this method, it is 242 even at 0.94 mU LAST / mL and 2018 TSAb%, 0.06 mU LAST / mL. It was found that detection was possible with TSAb%, and that the stimulation type antibody could be detected with about 16 times higher sensitivity than the existing kit (see FIG. 11).

12 Examination of time course of luminescence of the kit using frozen cells 1 ml of frozen cells prepared in 1 (3 × 10 ⁶ cells / ml) was dissolved in a warm bath and 10 mL of sample buffer (130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl ₂ , Suspended in 4.8 mM NaHCO ₃ , 5% PEG6000, pH 7.4), centrifuged at 1000 rpm for 5 minutes, added 10 mL of sample buffer to the resulting precipitate, added 7.5 μL 4 mM ViviRen (Promega), 90 μL / well Sowing in 96-well plates. After culturing at 37 ° C. CO ₂ for 3 hours, 10 μL of H (high value), M (medium value), and L (low value) samples purified with 30% PEG6000 were added (n = 6) and incubated for 30 minutes. 9 mM CaCl ₂ solution was added at 50 μL / well, and after stirring for 3 seconds, the amount of luminescence was measured with a luminometer over time for 13 seconds. As a result, as shown in FIG. 12, relatively stable luminescence was observed immediately after the addition of the CaCl ₂ solution.

13. Examination of quantitative properties of inhibitory antibody (TSBAb) against TSHR (1) Method of detecting inhibitory antibody (TSBAb) against TSHR using competitive inhibition with bTSH 1 ml of frozen cells prepared in 1 (3 × 10 ⁶ cells / ml) ) Was dissolved in a warm bath and suspended in 10 mL of sample buffer (130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl ₂ , 4.8 mM NaHCO ₃ , 5% PEG6000, pH 7.4) and obtained after centrifugation at 1000 rpm for 5 minutes. Add 10mL sample buffer to the precipitate, add 7.5μL 4mM ViviRen (Promega), incubate at 37 ° C CO ₂ for 3 hours, then dilute the inhibitory antibody from hypofunction patient purified with 30% PEG6000 After 10 μL of the series and 90 μL of the cell solution were mixed and incubated for 30 minutes, 9 mM CaCl ₂ solution was added at 50 μL / well, and the luminescence was measured with a luminometer.
As shown in FIG. 13, an inhibitory antibody (TSBAb) against TSHR was quantitatively detected using this frozen cell.
(2) Concentration dependence of the method for detecting an inhibitory antibody against TSHR (TSBAb) using cell desensitization 1 ml of frozen cells prepared in 1 (3 × 10 ⁶ cells / ml) were lysed in a warm bath and a 10 mL sample buffer was suspended in (3mM CaCl2, 130mM NaCl, 5mM KCl, 20mM HEPES, 1mM MgCl 2, 4.8mM NaHCO 3, 5% PEG6000, pH 7.4), after 1000 rpm 5 minutes centrifugation, the resulting 10mL of sample buffer to the precipitate 7.5 μL 4 mM ViviRen (Promega) was added and seeded in a 96-well plate at 90 μL / well. 10 μL of a sample series diluted with an inhibitory antibody derived from a hypofunction patient purified with 30% PEG6000 was added, and 10 μL / well of bTSH solution was added to a final concentration of 100 μL / mL, followed by 37 ° C. CO ₂ Then, 10 ⁻⁴ M forskolin solution was added at 50 μL / well and the amount of luminescence was measured with a luminometer.
As shown in FIG. 14, using this frozen cell, an inhibitory antibody against TSHR (TSBAb) was detected in a concentration-dependent manner.

14 Examination of action time (culture time) of stimulating antibody (TSAb) or inhibitory antibody (TSBAb) against TSHR (1) Method for detecting stimulating antibody (TSAb) 1 ml of frozen cells prepared in 1 (3 × 10 ⁶ cells / ml) ) Was dissolved in a warm bath and suspended in 10 mL of sample buffer (130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl ₂ , 4.8 mM NaHCO ₃ , 5% PEG6000, pH 7.4) and obtained after centrifugation at 1000 rpm for 5 minutes. To the precipitate, 10 mL of sample buffer was added, 7.5 μL 4 mM ViviRen (Promega) was added, and seeded in a 96-well plate at 90 μL / well. Add 10 μL of high (H), medium (M), low (L), normal (N) serum samples purified with 30% PEG6000, culture for 30 minutes to 1.5 hours, then add 9 mM CaCl ₂ solution to 50 μL / well And the amount of luminescence was measured with a luminometer.
As shown in FIG. 15, the action time of the cells according to the present invention and the stimulating antibody (TSAb) was 30 minutes to 1 hour, and the amount of luminescence reached a plateau.
(2) Examination of the action time in the method of detecting an inhibitory antibody (TSBAb) against TSHR using competitive inhibition with bTSH 1 ml of frozen cells prepared in 1 (3 × 10 ⁶ cells / ml) was lysed in a warm bath and 10 mL Suspend in sample buffer (130 mM NaCl, 5 mM KCl, 20 mM HEPES, 1 mM MgCl ₂ , 4.8 mM NaHCO ₃ , 5% PEG6000, pH 7.4), centrifuge at 1000 rpm for 5 minutes, add 10 mL of sample buffer to the resulting precipitate. 7.5 μL 4 mM ViviRen (Promega) was added, seeded in a 96-well plate at 90 μL / well, and cultured for 3 hours. Add 10 μL of serum samples from hypofunction patients purified with 30% PEG6000 and normal human serum samples, add 10 μL / well bTSH solution to a final concentration of 10 μU / mL, and incubate for 10 minutes to 2 hours Then, 9 mM CaCl ₂ solution was added at 50 μL / well, and the luminescence was measured with a luminometer.
As shown in FIG. 16, the action time of bTSH competing with the cell according to the present invention and the inhibitory antibody (TSBAb) was 30 minutes, and the amount of luminescence reached a plateau.
(3) Method for detecting an inhibitory antibody (TSBAb) against TSHR using cell desensitization 1 ml of frozen cells prepared in 1 (3 × 10 ⁶ cells / ml) was lysed in a warm bath and 10 mL of CO ₂ independent medium ( Cat No. 18045, Invitrogen) and centrifuged at 1000 rpm for 5 minutes. To the obtained precipitate, 7.5 mL of 4 mM ViviRen (Promega) was added in addition to 10 mL of CO ₂ independent medium, and 96 μg at 90 μL / well. It was seeded in a well plate and cultured for 3 hours. Add 10 μL of serum samples from hypofunction patients purified with 30% PEG6000 and normal human serum samples, add 10 μL / well bTSH solution to a final concentration of 100 μU / mL, and incubate for 10 minutes to 2 hours Then, 10-4 M forskolin solution was added at 50 μL / well, and the amount of luminescence was measured with a luminometer.
As shown in FIG. 17, the action time of bTSH competing with the cell according to the present invention and the inhibitory antibody (TSBAb) was 10 minutes, and the amount of luminescence reached a plateau.

15. Provided by the present invention is a frozen cell prepared in Measurement 1 in various thyroid disease patient samples , and a cell culture solution, cell washing solution, ViviRen, calcium solution, goat anti-hTSH antibody, control serum and 96-well plate described below. Using the Grace's disease diagnostic kit, we examined serum TSAb activity in patients with various thyroid diseases. Specifically, 198 untreated Graves' disease patients, 18 patients with hypofunction, 48 healthy people, 2 functional nodules, 6 postpartum thyroiditis, 6 painless thyroid Sera of 22 cases of inflammation and 22 cases of subacute thyroiditis were used (see Table 2). To 50 μL of patient serum, 150 μL of 30% PEG6000 was added and stirred, and then allowed to stand at 4 ° C. for 5 minutes. After centrifuging at 4 ° C., 3000 rpm for 20 minutes, the resulting precipitate was added to 400 μL of cell culture solution (5% PEG6000, CO ₂ -independent medium (calcium chloride, D-calcium pantothenate, L-glutamine, phenolred free, Invitrogen)) To give a sample solution. The sample solution was added to the 96-well plate at 10 μL / well (n = 2). 1 ml of frozen cells (3 × 10 ⁶ cells / ml) was dissolved in a warm bath and suspended in 10 ml of cell washing solution (CO ₂ independent medium). After centrifugation at 1000 rpm for 5 minutes, 12 mL of cell culture solution was added to the resulting precipitate to prepare a cell solution. 7.5 μL of 4 mM ViviRen was added to the cell solution, and seeded in a 96-well plate at 90 μL / well. After culturing for 4 hours, 100 μL of a 3 mM calcium solution was added, and the amount of luminescence was measured with a luminometer. Since some patients with hypofunction have high serum hTSH levels, goat anti-hTSH polyclonal antibody (Life Sciences) 1 is added to the cell culture solution to neutralize the action of hTSH. Measurement was performed under the condition that 2 μL was added. The measured value in each patient was calculated by a one-point calibration curve where the measured value of the control serum obtained by diluting NIBSC 65/122 with normal serum (1.874 mU LATS / mL) was 1800. The same patient serum was measured using an existing kit (TSAb kit, Yamasa) according to the kit attachment.
A cut-off of the TSAb value was set from the serum TSAb value of healthy individuals. FIG. 20 shows the frequency distribution of serum TSAb values in 48 healthy subjects. The average value of healthy people showed a normal distribution of 124 ± 34. The average value + 3SD = 180 was set as the cutoff.
The TSAb value of each thyroid disease is shown in FIG. In untreated Graves' disease, 195 out of 198 were positive (98%), in hypofunction three were positive, indolent thyroiditis, and one in postnatal thyroiditis was positive. In Table 3, the positive rate of Graves 'disease with the existing kit was examined in 198 patients with untreated Graves' disease. Since the positive rate of the existing kit was 68% and the kit provided by the present invention was 98%, it was found that the detection sensitivity of Graves' disease was significantly improved as compared with the existing kit.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to provide the measuring method and measuring kit of a TSH receptor antibody which are easy to operate and safe, and can diagnose a thyroid disease.

    The application that is the basis of the priority claim of this application is Japanese Patent Application: Japanese Patent Application No. 2009-155183, the entire contents of which are included in this application by reference.

Claims (6)

Thyroid disease using a biological sample derived from a subject, comprising a cell expressing a thyrotropin receptor (TSHR), a cAMP-dependent calcium channel, and a calcium-sensitive protein that is a protein that emits light in response to calcium. Diagnostic kit. Thyroid-stimulating hormone receptor (TSHR), cAMP-dependent calcium channel that is more sensitive to cAMP than cGMP, and cells expressing calcium-sensitive protein, a protein that emits light in response to calcium, anti-TSH antibody and / or TSHR A diagnostic kit for thyroid disease using a biological sample derived from a subject, comprising a stimulating antibody.   The cAMP-dependent calcium channel is a CNG calcium channel derived from murine olfactory epithelial cells, which is a modified CNG calcium channel comprising substitution of 460th cysteine with tryptophan and 583th glutamic acid with methionine. The kit in any one of 1-2.   TSHR is a TSHR having the amino acid sequence represented by SEQ ID NO: 1, the cAMP-dependent calcium channel is a modified CNG calcium channel having the amino acid sequence represented by SEQ ID NO: 2, and the calcium sensitive protein is SEQ ID NO: 3. The kit according to any one of claims 1 to 3, which is a modified apoaequorin having the amino acid sequence shown.   The kit according to any one of claims 1 to 4, wherein the thyroid disease is hyperthyroidism or hypothyroidism.   The kit according to any one of claims 1 to 4, wherein the thyroid disease is Graves' disease and / or Hashimoto's disease.